RESEARCH ARTICLE Open Access

Relaxin inhibits patellar tendon healing inrats: a histological and biochemicalevaluationTianpeng Xu1†, Jiaxiang Bai2†, Menglei Xu1†, Binqing Yu2, Jiayi Lin2, Xiaobin Guo2, Yu Liu2, Di Zhang1, Kai Yan1,Dan Hu1, Yuefeng Hao1* and Dechun Geng2*

Abstract

Background: Female patients are more likely to have tendon injuries than males, especially those who has a higherconcentration of relaxin. Previous studies have demonstrated that relaxin attenuates extracellular matrix (ECM)formation. However, the mechanism of relaxin on tendon repair remains unclear. We hypothesize that relaxininhibits tendon healing by disrupting collagen synthesis.

Methods: A patellar tendon window defect model was established using Sprague-Dawley rats. The center of thepatellar tendon was removed from the patella distal apex and inserted to the tibia tuberosity in width of 1 mm.Then, the rats were injected with saline (0.2 μg/kg/day) or relaxin (0.2 μg/kg/day) for two and four weeks, whichwas followed by biomechanical analysis and histological and histochemical examination.

Results: Mechanical results indicated that relaxin induces a significant decrease in tear resistance, stiffness, andYoung’s modulus compared to those rats without relaxin treatment. In addition, it was shown that relaxin activatesrelaxin family peptide receptor 1(RXFP1), disturbs the balance between matrix metalloproteinases (MMPs) and tissueinhibitors of metalloproteases (TIMPs), and reduces the deposition of collagen in injury areas.

Conclusions: Relaxin impairs tendon healing in rats. Also, relaxin might lead to tendon injury more commonly forfemales than males.

Keywords: Relaxin, Female, Tendon healing, Extracellular matrix (ECM), Relaxin family peptide receptor 1(RXFP1)

BackgroundTendons, which are dense fibrous connective tissuescomposed of tenocytes and abundant ECM, connectmuscles to bones and facilitate in the transmission ofmuscle-generated forces to the bones, resulting in jointmotion [1]. With the increasing popularity of sports,more people now engage in physical exercise. However,due to inappropriate actions, accidents, and populationaging, the morbidity of activity-related injuries such astendon injuries has rapidly increased. There are morethan 30 million tendon injuries around the world each

year, and the actual number is even higher because manyinjuries are not reported [2]. Unfortunately, tendonshave poor self-repair capability due to their nature ofminimal blood supply, poor oxygen consumption, andlow metabolic capacity [3]. Rui et al. showed that tendonhealing is a failed healing or nonhealing process. Histo-logically, tendinopathic tissues exhibit a failed healingcondition that is characterized by tissue metaplasia andinclude chondrocyte phenotypes, fatty infiltration, andbony deposits in some tendinopathy patients and animalmodels [4]. One of the important factors of tendon heal-ing was ECM. Changes in the physiological conditions ofECM after tendon injury may trigger protease activitiesand impair the balance between MMPs and TIMPs. Thisimbalance further induces collagen degeneration, whichis critical to tendon tissue integrity [5].

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

* Correspondence: 13913109339@163.com; szgengdc@163.com†Tianpeng Xu, Jiaxiang Bai and Menglei Xu contributed equally to this work.1Orthopedics and Sports Medicine Center, The Affiliated Suzhou Hospital ofNanjing Medical University, 242, Guangji Road, Suzhou 215006, People’sRepublic of China2Department of Orthopedics, The First Affiliated Hospital of SoochowUniversity, 188, shi zi Road, Suzhou 215006, People’s Republic of China

Xu et al. BMC Musculoskeletal Disorders (2019) 20:349 https://doi.org/10.1186/s12891-019-2729-3

Research investigations on sex hormones have re-cently increased. There is a quiet difference betweenwomen and men for the risk of tendon and ligamentinjuries. Sex hormonal differences may one of themajor reasons [6]. For example, estrogen had anegative effect on tendon healing and metabolism andreduced tendons composition like collagen I, aggrecanand elastin. Collagen I, aggrecan and elastin [6, 7].Meanwhile, Relaxin, a polypeptide hormone thatconsists of relaxin 1–3 and insulin-like peptides(INSL3, 4, 5, and 6), can also affect the tendons orligaments. The corpus luteum and the placentamainly synthesized it. Relaxin affects the extracellularmatrix components of tissues. The two main relaxinreceptor isoforms include RXFP1 and RXFP2. WhileH1 and H2 (human relaxin) activate RXFP1 andRXFP2, rat relaxin 1 weakly binds to RXFP2 [8].When mice are pregnant, relaxin transforms the pubic

articular cartilage to a pliable interligament in adapt to theenlarged uterus. Meanwhile, relaxin itself or combinedwith estrogen reduce public collagen content, which is an-tagonized by progesterone, in non-pregnant rats [9].It has been shown that relaxin is antifibrotic as it

downregulate fibroblast activity, increase collagenasesynthesis, and inhibits collagen-1 lattice contraction inrat kidneys, which is stimulated by transforming growthfactor-β [10]. According to Wang et al. [11], they findthat relaxin receptors are expressed in mouse knee fibro-cartilage, which suggest the knee is also a potential tar-get of relaxin. Relaxin receptors have been identifiedboth in females and males’ anterior cruciate ligament(ACL), meanwhile there is a high expression in females.Dehghan et al. [9] reported that RXFP1 and RXFP2 areexpressed in rat patellar tendon, which are upregulatedby progesterone and high doses of estrogen.Dragoo et al. [12] showed that female athletes

occurred with ACL tears have higher concentrationof relaxin. Those with serum relaxin concentrations> 6 pg/mL have a four-fold higher risk for tears.Athletes often develop chronic tendinopathy due tochronic tendon injuries or overuse of their tendonscoupled with insufficient rest after injuries. There-fore, the probability of ACL or other tendon tears issignificantly higher than in non-athletes. Duringsports activities, female athletes tear their ACL twoto eight times more frequently than males and thismay be attributable to relaxin, which influences thecomposition of ECM and contributes to tendondegeneration and increased risk of tendon injury infemale athletes [13, 14].However, the effect of relaxin on tendon and ligament

healing remains unclear. We hypothesized that relaxininhibits tendon healing, and carried out this study to testit in a rat patellar tendon window defect model.

MethodsAll animal procedures were approved by the Ethics Com-mittee of the First Affiliated Hospital of Soochow Univer-sity (Suzhou, Jiangsu, China).

AnimalsA total of 36 female Sprague-Dawley rats, 6–8 weeks oldand weighing from 250 to 300 g, were used in this study.The rats were purchased from the Experimental AnimalCenter of Soochow University. Also, they were kept in aventilated environment with a 12 h:12 h light-dark cycleat a constant temperature of 21 °C.

Rat patellar tendon repair modelTo induce tendon defects, two stacked sharp blades, de-scribed in a previous study, was used to remove the cen-ter of the patellar tendon (1 mm in width) from thepatella distal apex and insert to the tibia tuberosity with-out causing any damage to the fibrocartilage zone [15](Fig. 1). The surgery was performed under sterile condi-tions. After surgery, nylon 5–0 suture were used to closethe skin incision. The rats were placed on heating padsin the recovery cages until they recovered fromanesthesia. For the first 24 h of recovery, the animalswere housed in individual cages and were provided withsoftened rat chow. The operated rats were divided intotwo groups: the vehicle group and the relaxin group,with 12 rats in each group. The 12 rats that underwentsham surgery were used as the control group. The ve-hicle group received subcutaneous injection near theknees of saline (0.2 μg/kg/day) for two and four weeks.The relaxin group also received subcutaneous injection

Fig. 1 A 1 × 4mm window defect was created in patellar tendonof rat

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of relaxin (0.2 μg/kg/day) (Protech, Nevada, USA) fortwo and four weeks. The injection needle was a kind ofmicro syringe (Gaoge, Shanghai, China). Diameter ofneedle is 0.5 mm. When we did the injections, twooperators performed at the same time, one operator wasin fixing the neck and limbs of the rats, and the otheroperator was responsible for the injection. At the timeof injection, the needle tip of the syringe was injectededwith an angle of 30°-50° and avoided damage tosurrounding skin and subcutaneous tissue. The dose ofrelaxin used in this study was based on previousinvestigations [16]. The rats were sacrificed by intraperi-toneal injection an over dose of sodium pentobarbital(150 mg/kg) after 2 and four weeks.

Biomechanical analysisThe right patellar tendons of rats, sacrificed 2 and 4weeks post-operatively, were kept at room temperaturein PBS-soaked gauze pads until testing within 1 h aftercollection. To carry out tensile testing of the tendons,the patella and the proximal tibia were fixed at eachclamp (5 mm in between two clamps) of a testing ma-chine (Instron, MA, USA). The samples were tested at aspeed of 1 mm/s until failure.

Histological and histochemical examinationFor histological analysis, the left patellar tendon wasfixed in 4% paraformaldehyde for one day. Afterembedding in paraffin, the samples were sectionedlongitudinally at a thickness of 6 μm, then deparaffinizedin xylene, washed, and hydrated with washing ethanol.The sections were processed for histological examinationusing hematoxylin-eosin (H&E) and Masson trichromestaining. To carry out the regenerating tissue qualityevaluation, we adopted a histological scoring system[12]. The histological analysis was examined independ-ently by two blinded observers. The scores of seven de-fined tissue regeneration criteria, which were describedby Stoll (Additional file 1: Table S1), were then recorded[17]. A score of 12 was given to an intact Achilles ten-don without any surgical operation.

The sections were de-waxed, hydrated and inactivated.Then the tissue were blocked with 5% normal goat serumafter antigens repairing and incubated at 4 °C overnightusing Col I (1:500), Col III (1:500) (Abcam, Shanghai,China), RXFP1 (1:200) (Biorbyt, California, USA), MMP1(1:500), MMP9 (1:500) (Abcam, Shanghai, China) andMMP13 (1:500) (Biorbyt) monoclonal antibodies. Finally,they were incubated with HRP-conjugated secondary anti-body (1:2,000) for 1 h. Sections were colored by incubatingwith the chromogen 3.3′-diaminobenzidine (DAB) tetra-hydrochloride and counterstained with hematoxylin.One-way ANOVA and post-hoc Bonferroni tests with

SPSS 20.0 were adopted to carry out statistical analysesof histological score and biomechanical testing. Statis-tical significance was assigned at p values less than 0.05.

ResultRelaxin disrupts tendon healingThere was no loss of specimens during the tests. The re-sults of mechanical testing showed that tissue repairafter tendon removal at each postoperative time point inthe vehicle and relaxin groups was worse than in thecontrol group (Table 1). At two weeks after surgery,maximum load, stiffness, and Young’s modulus in boththe vehicle and relaxin group were lower than in thecontrol group (p < 0.05). This observation suggests thatsurgical operations change the mechanical properties oftendons. The results demonstrate that the vehicle andrelaxin groups have similar mechanical properties duringthe earlier period of repair. However, the data obtainedfour weeks postoperatively indicated that the controlgroup exhibited significantly higher maximum load andYoung’s modulus (p < 0.05) than the relaxin group.These findings suggest that relaxin disrupts tendon heal-ing, whereas the control group exhibited regeneration.No statistical difference in tendon length and cross-sec-tional areas between the three groups after two and fourweeks was observed.

Relaxin reduces the deposition of collagen in injury areasH&E staining revealed delayed healing in the relaxingroup (Fig. 2a). Early collagen formation was seen

Table 1 Results of mechanical testing

Groups Length (mm) Area (mm2) Maximum load (N) Stiffness (N/mm) Young’s modulus (MPa)

Two-week Control 6.02 ± 0.65 4.17 ± 0.80 74.83 ± 5.87 39.93 ± 5.99 18.95 ± 1.73

Vehicle 6.09 ± 0.66 4.77 ± 0.39 41.91 ± 11.19a 20.77 ± 3.40a 9.20 ± 2.63a

Relaxin 5.09 ± 0.55 4.06 ± 0.83 39.48 ± 5.93a 14.71 ± 3.17a 5.42 ± 2.70a

Four-week Control 5.95 ± 0.94 3.88 ± 0.70 76.54 ± 6.41 40.12 ± 5.82 17.66 ± 2.25

Vehicle 6.12 ± 0.35 4.91 ± 0.78 65.90 ± 7.69 28.26 ± 7.81a 15.91 ± 3.77

Relaxin 5.71 ± 0.94 4.39 ± 0.56 50.57 ± 9.91a,b 21.05 ± 5.96a 9.39 ± 3.37a,b

a p < 0.05 significantly different from the control groupb p < 0.05 significantly different from the vehicle group

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two and four weeks after wounding in the vehicleand the relaxin groups, respectively. Two weeks afterwounding, both vehicle and relaxin groups showedhigh cell density in the wound areas. However, fourweeks after wounding, the vehicle group exhibited amore distinct cell arrangement than the vehiclegroup. Masson staining was utilized to evaluate colla-gen maturation levels. At week 2, there were morecollagen found in vehicle group than in relaxin group.At week 4, there was further deposition observed.(Fig. 2a). At each time point, intact tendons showedsignificant differences in histological scores comparedwith both study groups (p < 0.05). However, the re-laxin group presented lower histological scores (p <0.05) than the vehicle group, indicating that the re-laxin group exhibited poor tendon structural recovery(Fig. 2b). These findings indicate that relaxin impairscollagen deposition in the wound areas.At week 2 and 4 after surgery, immunohistochem-

istry staining of Col I and Col III were applied inthe new tendon tissues. The expressions of Col Iand Col III in the neo-tendon tissues were strongerin the vehicle group and more uniform at bothweeks 2 and 4. Compared to normal tendons, theexpressions of Col I and III were more intense, sug-gesting that more ECM was deposited into thewound areas during tendon healing (Fig. 2c). Therelative expression of Col I and Col III is shown inFig. 2d, demonstrating a much higher abundance ofCol III of vehicle group relative to relaxin group(p < 0.05).

Relaxin changes the balance between MMPs and TIMP1by activating RXFP1To investigate how relaxin alters collagen deposition inwound areas, we first evaluated the expression ofRXFP1, which is the receptor for relaxin. Immunohisto-chemical staining showed higher RXFP1 expression inthe wound areas of the relaxin group compared to thevehicle group (Fig. 3a). An increase in RXFP1 expressionin the wound areas was observed in the relaxin group(p < 0.05) (Fig. 3b). These results reveal that relaxin in-creases RXFP1 expression in injured tendons.MMPs play an important role in collagen metabolism.

We assessed the effects of relaxin on the expression ofMMPs and TIMP1. Immunohistochemical staining indi-cated that MMP9 and MMP13 were highly expressed inthe relaxin group (p < 0.05). However, no difference inMMP1 and TIMP1 expression between the two groupswas observed. Relaxin treatment effectively increased theexpression of MMP9 and MMP13. These findings indi-cate an alteration in the balance between MMPs andTIMP expression (Fig. 4), thereby resulting in worsehealing in the relaxin group compared to the vehiclegroup.

DiscussionRelaxin, a peptide hormone belonging to the insulinsuperfamily, is involved in the promotion of ECM re-modeling. The present study has shown that ratsinjected with relaxin exhibited inferior healing of injuredtendons.

Fig. 2 Histopathological and immunohistochemical findings of repaired tendons in the vehicle and relaxin groups. a: H&E and Masson staining.b: Histological scores at the 2nd and 4th postoperative weeks. c: Immunohistochemical staining of collagen I and collagen III in the tendonwound areas in the 2nd and 4th postoperative weeks. *p < 0.05 vs. native tendon, #p < 0.05 compared with vehicle group. (Scale bar = 200 μm). d:Relative expression levels collagen I and collagen III in relaxin group compared with vehicle group. *p < 0.05 compared with vehicle group

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We evaluated the effects of relaxin on the mechanicalproperties of tendon healing. No significant differences inlength and cross-sectional areas were observed among thevehicle, relaxin, and control groups. This finding may bedue to that the rats do not produce a large amount of scartissues in the patellar tendon during injury. However, inthe rat Achilles tendon injury model, alterations in cross-sectional area and length were observed that operationhad caused severe scar tissue and increased cross-sectionalareas after healing of achilles tendon [18]. The presentstudy confirmed that both vehicle and relaxin groups ex-hibit lower maximum load, stiffness, and Young’s moduluscompared to the controls. However, the vehicle groupshowed improvement at four weeks after surgery, whereasthe maximum load and Young’s modulus of the relaxingroup remained worse than the control group. These

results demonstrate that relaxin disrupts the healingprocess, thereby resulting in weak tendons.The histological scores of the present rat model were indi-

cative of the degeneration of tendon mechanical propertiestwo or four weeks after surgical removal. H&E stainingshowed that relaxin inhibits tendon repair (Fig. 2b). Overtime, collagen deposition occurred in the wound area, as in-dicated by more intense Masson staining. Col I, essential forfibrous tissues repair and regeneration, is one the mainECM protein in tendon [19]. The collagen fibers longitu-dinal arrangement is important to provide mechanical prop-erties of tendons and can maintain the physiologicalstructure [20, 21]. The rat model showed that the controlgroup had more Col I and Col III accumulation in thewound area. All mechanical and histological data indicatethat repair tissues in the vehicle group, which did not

Fig. 4 Relaxin disrupts the balance between MMPs and TIMP. a: Immunohistochemical staining of TIMP1, MMP1, MMP9, and MMP13 in thetendon wound areas in the 2nd and 4th postoperative weeks. (Scale bar = 200 μm). b: Relative expression of MMP1, MMP9, MMP13 and TIMP1 intendon wound areas. MMP9 and MMP13 shows a significant difference in the 2nd and 4th postoperative weeks. *p < 0.001 compared withvehicle group

Fig. 3 Relaxin activates RXFP1 expression. a: Immunohistochemical staining of RXFP1 in the tendon wound areas in the 2nd and 4thpostoperative weeks. b: Quantification of RXPF1 positive cells expression in the tendon wound areas. *p < 0.001 compared with vehicle group.(Scale bar = 200 μm)

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receive relaxin treatment, exhibited more extensiveimprovement than the relaxin group.The MMPs family is composed of more than 20 zinc-

dependent metalloproteinases that are responsible for celladhesion, migration, proliferation, and differentiation [22].MMP1 and MMP13 are members of this family that arestrongly associated with collagen degradation and metab-olism [23]. In the present study, the expression of Col Iand Col III in tendon ECM was significantly higher in thecontrol group, whereas that of RXFP1 and MMP13 wasupregulated in the relaxin group. These findings revealthat relaxin stimulates the expression of MMP13 byactivating RXPF1, thereby altering the structure and pro-moting the degradation of collagen. These results implythat RXPF1 and MMPs expression in vivo may be utilizedas an indicator for the risk of ligament tears.Previous rat studies have shown that drugs,

exercise, and biomaterials improve tendon healing[24–27]. Moreover, the change of sex hormone arerelated to tendon diseases. Ganderton et al. foundthat menopausal hormone therapy (MHT) could im-prove the pain and function in post-menopausalwomen with greater trochanteric pain syndrome(GTPS) [28]. Furthermore, external factors can en-hance the histological and biomechanical propertiesof tendons. However, the regulatory mechanismunderlying the inhibition of tendon repair remainselusive. Zhang al et [29]. proposed that tendon heal-ing is a failure process because of poor blood supplyand nerve growth and the imbalance of ECM. Re-laxin can activate MMPs, which in turn promotecollagen degradation in the fibrocartilaginous tem-poromandibular joint [30]. Changes in the physio-logical conditions of the ECM following tendoninjury can trigger protease activity and compromisethe balance between MMPs and TIMP. This imbal-ance would further induce collagen degeneration,causing poor tendon and ligament healing.The findings of this study also showed that in rat

patellar tendons, relaxin increased the expression ofRXFP1 and promoted the expression of MMP9 and13, which in turn reduced collagen deposition in thewound area. In addition, the findings of this studyalso identify a novel target to improve the healing oftendons.One of the limitations of this study is that we did not in-

vestigate the effect of relaxin on tenocytes or tendon stemcells and the animal models did not include a castrationgroup and did not assess whether relaxin has the samebiological effects on castration. Secondly, it is not clearwhether downstream signaling pathways activate RXFP1.Lastly, we applied a patellar ligament injury model, whichis not happened in nature. This did not fully explain thehealing mechanism of human tendon ligament injury.

ConclusionRelaxin disrupts patellar tendon healing in a rat model.Further in vitro experiments confirming the effect of re-laxin on tenocytes or tendon stem cells are warranted.Although the levels of hormones in humans and rats arenot the same, women exhibit more extensive fluctua-tions in sex hormone levels during the menstrual cycle.In addition, studies on the role of relaxin in ligamenthealing should be performed.

Additional file

Additional file 1: Table S1. Histological scoring system. (DOCX 17 kb)

AbbreviationsACL H&E: Hematoxylin-eosin; ACL: Anterior cruciate ligament; DPX: p-xylene-bis-pyridinium bromide; ECM: Extracellular matrix; INSL: Insulin-like peptides;MMPs: Matrix metalloproteinases; one-way ANOVA: One-way Analysis ofVariance; PBS: Phosphate Buffered Saline; RXFP1: Relaxin family peptidereceptor; RXFP2: Relaxin family peptide receptor 2; TIMPs: Tissue inhibitors ofmetalloproteases

AcknowledgementsAt the point of finishing this paper, I’d like to express my sincere thanks toall those who have lent me hands while my writing this paper. I’d like toexpress my gratitude to Professor. Qin Shi who offered the site for ourresearches and Dr. Mimi Chen who helped design experiments and providedguidance

Authors’ contributionsDCG, YFH and TPX conceived and designed the research. JXB, MLX, TPX, JYLand KY performed the experiments, collected data and conducted research.XBG, YL, DH and BQY analyzed and interpreted data. TPX wrote the initialmanuscript. DCG, YFH and DZ revised the manuscript. DCG was primarilyresponsible for the final content. All authors have reviewed and approvedthe final manuscript.

FundingThis research was supported by the National Nature Science Foundation ofChina (81873990, 81873991, 81472077, 81672238 and 81372018), the NaturalScience Foundation of Jiangsu province (BK20180001), the Jiangsu ProvincialMedical Youth Talent (QNRC2016751), the Key Project Supported by theMedical Science and Technology Department of Foundation, JiangsuProvince, Department of Health (H2018027) and the ApplicationFundamental Research Program of Suzhou City (SYSD2016072). The NSFCand NSFJ provided the location for experiments and animal feeding. TheKPSMSTDF,TDF and AFRP provided the fund for design of the study andcollection, analysis, and interpretation of data. All of these funding supportedthe writing of the manuscript.

Availability of data and materialsThe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable request.

Ethics approval and consent to participateAnimal welfare and experimental procedures were carried out in accordancewith the Declaration of Helsinki, and were approved by the EthicsCommittee of the First Affiliated Hospital of Soochow University. (ApprovalNumber: 201805A008).

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

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Received: 15 August 2018 Accepted: 18 July 2019

References1. Öhberg L, Lorentzon R, Alfredson H. Eccentric training in patients with

chronic Achilles tendinosis: normalised tendon structure and decreasedthickness at follow up. Br J Sports Med. 2004;38(1):8–11.

2. Sharma P, Maffulli N. Tendon injury and tendinopathy: healing and repair.JBJS. 2005;87(1):187–202.

3. Sharma P, Maffulli N. Biology of tendon injury: healing, modeling andremodeling. J Musculoskelet Nueronal Interact. 2006;6(2):181.

4. Rui YF, Lui PPY, Wong YM, Tan Q, Chan KM. Altered fate of tendon-derivedstem cells isolated from a failed tendon-healing animal model oftendinopathy. Stem Cells Dev. 2012;22(7):1076–85.

5. Konopka JA, DeBaun MR, Chang W, Dragoo JL. The intracellular effect ofRelaxin on female anterior cruciate ligament cells. Am J Sports Med. 2016;44(9):2384–92.

6. Hansen M, Kjaer M. Sex hormones and tendon. In: Metabolic influences onrisk for tendon disorders, vol. 2016: Springer. p. 139–49.

7. Oliva F, Piccirilli E, Berardi AC, Frizziero A, Tarantino U, Maffulli N. Hormonesand tendinopathies: the current evidence. Br Med Bull. 2016;117(1).

8. Conrad KP, Baker VL. Corpus luteal contribution to maternal pregnancyphysiology and outcomes in assisted reproductive technologies. Am J PhysRegul Integr Comp Phys. 2012;304(2):R69–72.

9. Dehghan F, Muniandy S, Yusof A, Salleh N. Sex-steroid regulation of relaxinreceptor isoforms (RXFP1 & RXFP2) expression in the patellar tendon andlateral collateral ligament of female WKY rats. Int J Med Sci. 2014;11(2):180.

10. Sasser JM. The emerging role of relaxin as a novel therapeutic pathway inthe treatment of chronic kidney disease. Am J Phys Regul Integr CompPhys. 2013;305(6):R559–65.

11. Wang W, Hayami T, Kapila S. Female hormone receptors are differentiallyexpressed in mouse fibrocartilages. Osteoarthr Cartil. 2009;17(5):646–54.

12. Dragoo JL, Castillo TN, Braun HJ, Ridley BA, Kennedy AC, Golish SR.Prospective correlation between serum relaxin concentration and anteriorcruciate ligament tears among elite collegiate female athletes. Am J SportsMed. 2011;39(10):2175–80.

13. Gwinn DE, Wilckens JH, McDevitt ER, Ross G, Kao T-C. The relative incidenceof anterior cruciate ligament injury in men and women at the United Statesnaval academy. Am J Sports Med. 2000;28(1):98–102.

14. Arendt E, Dick R. Knee injury patterns among men and women in collegiatebasketball and soccer: NCAA data and review of literature. Am J Sports Med.1995;23(6):694–701.

15. Lui PP, Cheuk YC, Hung LK, Fu SC, Chan KM. Increased apoptosis at the latestage of tendon healing. Wound repair and regeneration : officialpublication of the Wound Healing Society [and] the European Tissue RepairSociety. 2007;15(5):702–7.

16. Cai J, Chen X, Chen X, Chen L, Zheng G, Zhou H, Zhou X. Anti-fibrosis effectof Relaxin and spironolactone combined on Isoprenaline-inducedmyocardial fibrosis in rats via inhibition of endothelial-mesenchymaltransition. Cell Physiol Biochem. 2017;41(3):1167–78.

17. Stoll C, John T, Conrad C, Lohan A, Hondke S, Ertel W, Kaps C, EndresM, Sittinger M, Ringe J. Healing parameters in a rabbit partial tendondefect following tenocyte/biomaterial implantation. Biomaterials. 2011;32(21):4806–15.

18. Muller SA, Evans CH, Heisterbach PE, Majewski M. The role of theParatenon in Achilles tendon healing: a study in rats. Am J Sports Med.2018;46(5):1214–9.

19. Masic A, Bertinetti L, Schuetz R, Chang S-W, Metzger TH, Buehler MJ, FratzlP. Osmotic pressure induced tensile forces in tendon collagen. NatCommun. 2015;6:5942.

20. Robinson KA, Sun M, Barnum CE, Weiss SN, Huegel J, Shetye SS, Lin L, SaezD, Adams SM, Iozzo RV. Decorin and biglycan are necessary for maintainingcollagen fibril structure, fiber realignment, and mechanical properties ofmature tendons. Matrix Biol. 2017;64:81–93.

21. Gilotra MN, Shorofsky MJ, Stein JA, Murthi AM. Healing of rotator cufftendons using botulinum toxin a and immobilization in a rat model. BMCMusculoskelet Disord. 2016;17:127.

22. Rohani MG, Parks WC. Matrix remodeling by MMPs during wound repair.Matrix Biol. 2015;44:113–21.

23. Salamanna F, Frizziero A, Pagani S, Giavaresi G, Curzi D, Falcieri E, Marini M,Abruzzo P, Martini L, Fini M. Metabolic and cytoprotective effects of in vivo

peri-patellar hyaluronic acid injections in cultured tenocytes. Connect TissueRes. 2015;56(1):35–43.

24. Jiang D, Gao P, Lin H, Geng H. Curcumin improves tendon healing in rats: ahistological, biochemical, and functional evaluation. Connect Tissue Res.2016;57(1):20–7.

25. Zhang J, Yuan T, Wang JH. Moderate treadmill running exercise priorto tendon injury enhances wound healing in aging rats. Oncotarget.2016;7(8):8498.

26. Xu Y, Dong S, Zhou Q, Mo X, Song L, Hou T, Wu J, Li S, Li Y, Li P, et al. Theeffect of mechanical stimulation on the maturation of TDSCs-poly(L-lactide-co-e-caprolactone)/collagen scaffold constructs for tendon tissueengineering. Biomaterials. 2014;35(9):2760–72.

27. Kraus TM, Imhoff FB, Reinert J, Wexel G, Wolf A, Hirsch D, Hofmann A,Stockle U, Buchmann S, Tischer T, et al. Stem cells and bFGF in tendonhealing: effects of lentiviral gene transfer and long-term follow-up in a ratAchilles tendon defect model. BMC Musculoskelet Disord. 2016;17:148.

28. Ganderton C, Semciw A, Cook J, Pizzari T. Does menopausal hormonetherapy (MHT), exercise or a combination of both, improve pain andfunction in post-menopausal women with greater trochanteric painsyndrome (GTPS)? A randomised controlled trial. BMC Womens Health.2016;16:32.

29. Zhang X, Y-c L, Y-f R, H-l X, Chen H, Wang C, G-j T. Therapeutic roles oftendon stem/progenitor cells in tendinopathy. Stem Cells Int. 2016;2016.

30. Shahin AKA, Sharrawy EAEHE, El AE-DSA, Hamed TAEB. The role of RELAXINhormone in internal derangement of TEMPROMANDIBULAR joint. ZagazigUniversity Medical Journal. 2015;19:5.

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